How many duodenal biopsy specimens are required to make a diagnosis of celiac disease?

ORIGINAL ARTICLE: Clinical Endoscopy

How many duodenal biopsy specimens are required to make a diagnosis of celiac disease? Wilson P. Pais, MD, Donald R. Duerksen, MD, Norman M. Pettigrew, MD, Charles N. Bernstein, MD Winnipeg, Manitoba, Canada

Background: It is unknown how many endoscopic biopsy specimens are needed to diagnose celiac disease (CD). Objective: To determine the numbers of duodenal biopsy specimens needed to diagnose CD. Design: Retrospective medical record audit, histology slide analysis, and chart review. Setting: A tertiary-care university hospital. Patients: Adults who underwent EGD to diagnose CD. Interventions: Our pathology database was searched for the keywords ‘‘consistent with celiac disease,’’ ‘‘not consistent with celiac disease,’’ ‘‘villous atrophy,’’ and ‘‘intraepithelial lymphocytes’’ from January 2001 to May 2006. The number of biopsy specimens was determined and graded for a modified Marsh classification, and charts were reviewed for diagnosis verification. CD was confirmed if Marsh grade 3A was found, even on one biopsy specimen. Main Outcome Measurements: The number of biopsy specimens needed to make the diagnosis of CD. Results: Of 247 cases, 102 patients were diagnosed with biopsy specimen–confirmed CD. In 9 patients, CD could not be confirmed on the basis of histology alone (highest Marsh lesion was grade 1 or 2), but a clinical diagnosis was made on the basis of presentation and serology. CD could be confirmed if only 2 biopsy specimens were obtained in 84 patients (90%), if only 3 biopsy specimens were obtained in an additional 5 patients (95%), and if at least 4 biopsy specimens were taken in the remainder. CD was ruled out in 145 patients. In 142 patients, biopsy specimens were uniformly negative; 3 patients had Marsh grade 1 lesions but negative serology. Limitations: A retrospective design. Conclusions: Only 2 biopsy specimens will lead to a confirmed diagnosis of CD in 90%, and a suspected diagnosis in all. For 100% confidence in diagnosis of CD, 4 duodenal biopsy specimens should be taken. (Gastrointest Endosc 2008;67:1082-7.)

Celiac disease (CD) is an autoimmune disorder of smallintestine inflammation induced by gluten proteins contained in wheat, barley, and rye.1 CD occurs in genetically susceptible individuals who are carrying human leukocyte antigen (HLA) DQ2 and DQ8 haplotypes.2 CD is more common than previously thought, with a suspected prevalence in the United States of 1:133.3 CD can present with a variety of symptoms, such as abdominal pain, abdominal

Abbreviations: CD, celiac disease; EMA, endomysial antibody; HLA, human leukocyte antigen; tTG, tissue transglutaminase. Copyright ª 2008 by the American Society for Gastrointestinal Endoscopy 0016-5107/$32.00 doi:10.1016/j.gie.2007.10.015

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distension, diarrhea, or weakness, or even among asymptomatic persons with signs of iron deficiency anemia or osteoporosis. In fact, it is becoming increasingly more common for CD to be diagnosed because of asymptomatic screening of subjects at risk, such as those with diabetes mellitus type I or first-degree relatives of affected persons. Antibodies to tissue transglutaminase (tTG) and the endomysial antibody (EMA) are highly effective screening tests and contribute to the diagnosis when a small-bowel biopsy specimen does not confirm the diagnosis with certainty. These serologic tests carry a high sensitivity and specificity.4 Despite these high sensitivities and specificities, current recommendations are to confirm the diagnosis of CD with a small-bowel biopsy specimen.5 Although abnormal small-bowel biopsy specimens are not specific www.giejournal.org

Pais et al

for CD, in the appropriate clinical setting, abnormal biopsy specimens confirm the diagnosis.6 There is a spectrum of histologic abnormalities found in CD, and these have been characterized by Marsh and modified by Oberhuber.7 The classic lesion is the destructive lesion or Marsh type 3 lesion, whereas the Marsh type 1 (intraepithelial lymphocytosis) and the Marsh type 2 (crypt proliferation) are more subtle. It is believed that the small-bowel mucosal damage is a dynamic process and has 3 phases. In phase 1, the infiltrative phase, there will be increased numbers of intraepithelial lymphocytes. In phase 2, the hyperplastic phase, there will be crypt hypertrophy, and in phase 3, the destructive phase, there will be villous atrophy.8 The mucosal lesions in CD can be patchy in children and adults,9,10 and, therefore, there is the possibility of missing the diagnosis if there is insufficient sampling. It is unknown how many biopsy specimens are needed to confirm the diagnosis of CD. A guideline from the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition made no recommendation as to the requisite number of biopsy specimens,11 whereas, ideally 6 biopsy specimens have been recommended by the American Gastroenterology Association.5 A false-positive or a false-negative diagnosis has a significant impact, because the only available therapy is lifelong abstinence from wheat and other cereals that contain gluten. The purpose of this study was to determine the number of biopsy specimens needed to confirm the diagnosis of CD, while keeping in mind that the histologic distribution of lesions can be patchy in CD. It has been our practice to obtain at least 4 random biopsy specimens from the second part of the duodenum to confirm or exclude the diagnosis of adult CD. However, there exists a considerable variation in this practice, including in our own center. In this study, we aimed to determine the minimum number of biopsy specimens required to confirm a diagnosis of CD by histology. We also wanted to determine the variation in histology in individual patients and the value of a completely normal small-bowel biopsy specimen.

MATERIALS AND METHODS All results in the Health Sciences Centre (a tertiary-care university hospital) Department of Pathology biopsy database from January 2001 to May 2006 were searched with the keywords ‘‘consistent with celiac disease,’’ ‘‘not consistent with celiac disease,’’ ‘‘villous atrophy,’’ and ‘‘intraepithelial lymphocytes.’’ This was intended to identify subjects in whom a diagnosis of CD was being sought by an EGD and a biopsy. A chart audit was carried out for all subjects identified. All the pathology slides were retrieved, the number of biopsy specimens was determined, and each specimen was graded according to the Marsh classification modified by Oberhuber.7 All biopsy speciwww.giejournal.org

Diagnosis of celiac disease

Capsule Summary What is already known on this topic d

Mucosal lesions in the small bowel can be patchy, so insufficient sampling can miss the diagnosis of celiac disease (CD).

What this study adds to our knowledge d

In a retrospective review of 247 adults who underwent endoscopy to diagnose CD, the diagnosis could have been confirmed in 90% if only 2 biopsy specimens were taken; a third biopsy led to confirmation in an additional 5%.

mens were assessed and graded by a single investigator (W.P.), and, in instances of uncertainty, the grading was undertaken by a second investigator (N.P.), and consensus was reached (see Table 1 for classification used7 and Fig. 1 A to E for representative examples). In keeping with the recently published guidelines of the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition, we considered Marsh grade 3A or higher to confirm a diagnosis of CD, even if found in only one biopsy specimen. Although Marsh type 1 and 2 lesions may also be consistent with CD, the findings are more subtle and could be associated with other conditions.12 The final diagnosis of CD was established after a chart review by considering clinical symptoms, signs, and serologic testing, together with biopsy specimen results. The serologic data available were incomplete in our study because of the retrospective nature and variable practice among gastroenterologists in ordering serology. At our center, it is a common but not uniform practice to obtain 4 biopsy specimens from the duodenum when CD is in question. A standard disposable endoscopy forceps is used, and biopsy specimens are obtained in the second or third part of the duodenum. Biopsy specimens are not mounted and are immediately placed in formalin. For the cases that were ultimately determined to be classifiable as CD based on the constellation of clinical findings, including biopsy specimens, we determined how many biopsy specimens would be needed to confirm the diagnosis solely based on biopsy specimens. We arranged the specimens in ascending order of a Marsh grade lesion and determined how many specimens were needed to reach Marsh grade 3A or higher lesion.

RESULTS Of the 248 patients identified, 1 patient was excluded because of damaged biopsy specimens. Of the remaining 247 patients, 171 were women (mean age 49.25 years) Volume 67, No. 7 : 2008 GASTROINTESTINAL ENDOSCOPY 1083

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TABLE 1. Classification of duodenal histologic lesions in CD* Grade 0

1

2

3a

3b

3c

!40

O40

O40

O40

O40

O40

Crypts

Normal

Normal

Hypertrophic

Hypertrophic

Hypertrophic

Hypertrophic

Villi

Normal

Normal

Normal

Mild atrophy

Marked atrophy

Absent

IEL count

IEL count, Intraepithelial lymphocytes per 100 epithelial cells. *From Ref. 7.

and 76 were men (mean age 51.42 years) (range [for all 247 patients] 17-88 years). Of these 247 patients, 102 were diagnosed with biopsy specimen–confirmed CD (29 men with a mean age of 53.68 years and 73 women with a mean age of 50.75 years). CD has a female preponderance, with a female to male ratio of 2:1 or 3:1.13-15 In a large North American study, patients with CD were predominantly female, with a female to male ratio of 2.1:1.16 Similar findings were noted in our study as well. The number of biopsy specimens taken per case in CD were as follows: 1 in 1 case, 2 in 13 cases, 3 in 22 cases, and at least 4 in 66 cases. All 102 cases had abnormal biopsy specimens, with at least Marsh grade 1 lesions in all biopsy specimens. In addition, the total number of biopsy specimens for the CD group was 399 (for 102 patients), and the average number of biopsy specimens per patient was 3.87. There were 9 cases in which CD could not be confirmed on the basis of histology alone (the highest Marsh lesion identified was grade 1 or 2). Of the remaining 93 cases, CD was confirmed with at least one biopsy specimen being modified Marsh grade 3A or higher. We found 1 biopsy specimen that led to a positive diagnosis in 80 patients (86%) (that is, the first specimen was Marsh IIIA or higher, and none of the specimens were less than Marsh IIIA); 2 biopsy specimens led to a positive diagnosis in 84 patients (90%) (among the additional 4 patients, the first specimen was Marsh II or lower, and the second specimen was Marsh IIIA or higher); 3 biopsy specimens led to a positive diagnosis in 89 patients (95%) (among the additional 5 patients, the first and second specimens were Marsh II or lower, and the third specimen was Marsh IIIA or higher), and 4 biopsy specimens led to a positive diagnosis in 93 patients (100%) (among the additional 4 patients, the first, second, and third specimens were Marsh II or lower, and the fourth specimen was Marsh IIIA or higher). When biopsy specimens were compared in each patient, we found that 76 specimens (75%) had identical Marsh grade, 18 (18%) had 1 grade difference, and 8 (8%) had 2 or more grade differences. See Table 2 for the difference in histologic grades in patients with CD. The most frequently seen lesion was 3B in 47 of 102 patients with CD. See Table 3 for frequency of distribution of the highest grade lesions in patients with CD. In pa-

tients with CD, the most common main presenting symptoms were abdominal pain (44%), anemia (17%), and diarrhea (11%). We identified 145 subjects in whom CD was ruled out. There were 47 men (mean age 50.02 years) and 98 women (mean age 48.18 years). The number of biopsy specimens per case was the following: 1 in 1 case, 2 in 16 cases, 3 in 37 cases, 4 or more in 91 cases. We considered CD to be excluded if a modified Marsh histologic lesion of grade 0 was found in all biopsy specimens. The total number of biopsy specimens for the non-CD group was 548 (for 145 patients), and the average number of biopsy specimens per patient was 3.79. Hence, even in the non-CD group, there were nearly 4 biopsy specimens per subject. Of the 145 patients in the non-CD group, 142 had modified Marsh grade lesion 0 in all biopsy specimens and were conclusively negative for CD. In the remaining 3 patients, Marsh grade 1 lesions were identified, but antibody testing was negative, and the gastroenterologist established a diagnosis as ‘‘non-CD.’’ In the 17 cases in which only 1 or 2 biopsy specimens were taken, serology was negative in all. In patients with non-CD, the most common main presenting symptoms were abdominal pain (40%), anemia (28%), and diarrhea (17%). Of the 102 patients with CD, 69 had EMA testing, 60 of whom were positive (87%), and 83 had tTG testing, 67 of whom were positive (81%). The tTG titers were highest with a more severe histologic grade (see Table 4). Of the 145 patients who were non-CD, 10 had EMA testing, and all 10 of them were negative (100%); 78 of the 145 patients had tissue tTG testing, and 74 of them were negative (95%). All 17 patients with 1 or 2 biopsy specimens had negative serology. Among 4 positive tTG results, the average titer was 34.5 RU/mL (the range of positive measurement for tTG in our laboratory is 20-200 RU/mL). All 4 of these subjects were EMA negative.

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DISCUSSION Even though noninvasive antibody testing is an excellent screening tool for CD, it is still recommended that a smallbowel biopsy be performed in all individuals to confirm the diagnosis.5,11 A combination of antibody titer, symptoms,

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Diagnosis of celiac disease

Figure 1. A, Marsh I lesion (H&E, orig. mag. 100). B, Marsh II lesion (H&E, orig. mag. 100). C, Marsh 3a lesion (H&E, orig. mag. 100). D, Marsh 3b lesion (H&E, orig. mag. 100). E, Marsh 3c lesion (H&E, orig. mag. 100).

and histologic lesion according to modified Marsh grade 1 or 2 can help in diagnosis in questionable cases. Genotyping for HLA DQ2 and DQ8 has a low specificity, but, if found to be negative, can also help assist in excluding the diagnosis of CD.17 It was previously determined that the site of the biopsy within the second, third, or fourth portions of the www.giejournal.org

duodenum, or the use of a standard biopsy forceps versus jumbo biopsy forceps does not affect the quality of the specimen obtained in diagnosing CD.18 The histologic recovery of small-bowel lesions is slow and incomplete, and takes up to 2 years to resolve19; hence, even if the patient goes on a short duration of Volume 67, No. 7 : 2008 GASTROINTESTINAL ENDOSCOPY 1085

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TABLE 2. Difference in histologic grades found in patients with CD

TABLE 4. Histologic grade and corresponding titers of tTG antibodies

Difference in grade

Lesion grade

tTG Titer*

No. patients (n Z 102)

No difference

77

3c

129 RU/mL

1 grade difference

16

3b

127 RU/mL

2 grade difference

6

3a

109 RU/mL

3 grade difference

3

2

N/A

4 grade difference

0

1

8 RU/mL

5 grade difference

0

0

4 RU/mL

*tTG titer: 0-19 means negative for CD; 20-200 means positive for CD. Results are reported in numerical value up to 200, and values greater than 200 are reported as O200.

TABLE 3. Frequency of distribution of highest grade histologic lesions found in patients with CD

a gluten-free diet on a trial basis, the confidence to diagnose the disease remains excellent from duodenal histopathology. An endoscopic biopsy of the duodenum is associated with a low morbidity, and multiple biopsy specimens can be obtained at the time of the procedure. Obtaining multiple biopsy specimens adds minimal additional time to the procedure. Multiple biopsy specimens add some cost to the examination, because more pathology time is required to process and read the specimens. Currently, there are no clear recommendations as to the exact number of biopsy specimens that should be taken at the time of an EGD when CD is suspected, and this retrospective study confirmed that there is some variability in this practice. It is important to obtain sufficient biopsy material to be certain that a diagnosis has not been missed because of sampling error. However, unnecessary biopsy specimens simply add to the cost of the test and the workload of the pathologist. In this study, taking only 2 biopsy specimens led to the confirmation of the diagnosis in 90% of cases, whereas 4 biopsy specimens led to a diagnosis 100% of the time. Therefore, based on these data, we would recommend taking 4 random duodenal biopsy specimens when CD is suspected. This is an important diagnosis to make, because it implies a lifetime commitment to a gluten-free diet. Our data support the literature that suggests that CD can be patchy. In our study, 25% of duodenal biopsy

specimens demonstrated significant variability, with at least one Marsh grade difference between biopsy specimens. For the clinician, this reinforces the need to take multiple random duodenal biopsy specimens. In the cases where CD was ruled out, 100% of biopsy specimens in non-CD cases showed uniform findings. The major limitation of this study was that it was a retrospective analysis. To absolutely prove that 4 biopsy specimens would be both necessary and sufficient to confirm (or refute) a CD diagnosis, this would better be done prospectively, with longitudinal follow-up of all of those considered to be negative. Furthermore, data for antibody testing were not available in all cases. Hence, among the non-CD cases, it is possible that those cases with fewer than 4 biopsy specimens might have had CD-like lesions found if additional biopsy specimens had been taken. However, charts were reviewed on all subjects, and, ultimately, a diagnosis of CD was not made, including in those with even fewer than 4 biopsy specimens. Therefore, our recommendation regarding the need for 4 biopsy specimens mostly comes from those 102 cases in which CD was clinically diagnosed. An advantage of our study was that all of the biopsy specimens were reviewed by a single investigator according to Marsh criteria modified by Oberhuber, and Marsh type 3 lesions were considered to be confirmatory for the diagnosis. Furthermore, our study was conducted in a unit where biopsy specimens are not mounted before submission for histopathologic review. This reflects the practice in the majority of endoscopy units in North America, which provides a measure of reassurance for those units where mounting is not undertaken. We, therefore, believe that the results are of value for the practicing clinician. In summary, our study suggests that there is histologic variability in CD and that, for unmounted specimens, 4 random duodenal biopsy specimens should be taken to confirm the diagnosis of CD in suspected cases.

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Highest grade lesion

No. patients (n Z 102)

Grade 0

0

Grade 1

8

Grade 2

1

Grade 3a

19

Grade 3b

47

Grade 3c

27

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DISCLOSURE The authors report that there are no disclosures relevant to this publication. C. Bernstein is supported in part by a Crohn’s and Colitis Foundation of Canada Research Scientist Award.

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Diagnosis of celiac disease 11. Hill ID, Dirks MH, Liptak GS, et al. Guideline for the diagnosis and treatment of celiac disease in children: recommendations of the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition. J Pediatr Gastroenterol Nutr 2005;40:1-19. 12. Chang F, Mahadeva U, Deere H. Pathological and clinical significance of increased intraepithelial lymphocytes (IELs) in small bowel mucosa. APMIS 2005;113:385-99. 13. Bai D, Brar P, Holleran S, et al. Effect of gender on the manifestations of celiac disease: evidence for greater malabsorption in men. Scand J Gastroenterol 2005;40:183-7. 14. Ciacci C, Cirillo M, Sollazzo R, et al. Gender and clinical presentation in adult celiac disease. Scand J Gastroenterol 1995;30:1077-81. 15. American Gastroenterological Association medical position statement: celiac sprue. Gastroenterology 2001;120:1522-5. 16. Rampertab SD, Pooran N, Brar P, et al. Trends in the presentation of celiac disease. Am J Med 2006;119:355, e9-14. 17. Kaukinen K, Partanen J, Maki M, et al. HLA-DQ typing in the diagnosis of celiac disease. Am J Gastroenterol 2002;97:695-9. 18. Dandalides SM, Carey WD, Peters R, et al. Endoscopic small bowel mucosal biopsy: a controlled trial evaluating forceps size and biopsy location in the diagnosis of normal and abnormal mucosal architecture. Gastrointest Endosc 1989;35:197-200. 19. Wahab PJ, Meijer JWR, Mulder CJJ. Histologic follow-up of people with celiac disease on a gluten-free diet: slow and incomplete recovery. Am J Clin Pathol 2002;118:459-63.

Received April 8, 2007. Accepted October 3, 2007. Departments of Internal Medicine (W.P.P., D.R.D., C.N.B.) and Pathology (N.M.P.), University of Manitoba, the University of Manitoba Inflammatory Bowel Disease Clinical and Research Centre (C.N.B.), Winnipeg, Manitoba, Canada. Reprint requests: Charles N. Bernstein, MD, Department of Medicine/ Gastroenterology, University of Manitoba 804F-715 McDermot Ave, Winnipeg, Manitoba, Canada R3E 3P4.

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